Saccharomyces-specific antigens and their use

ABSTRACT

Antibodies which are specific for Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces paradoxus and Saccharomyces pastorianus, and epitopes to which such antibodies bind are described. Diagnostic agents and assays for detecting Saccharomyces sensu strictu and epitopes of Saccharomyces sensu strictu also are described.

This application is a divisional of application Ser. No. 08/377,737,filed Jan. 25, 1995, now U.S. Pat. No. 5,718,898.

FIELD OF THE INVENTION

The invention relates to antibodies which are specific for Saccharomycescerevisiae, Saccharomyces bayanus, Saccharomyces paradoxes andSaccharomyces pastorianus, and to epitopes to which such antibodiesbind. These antigens and antibodies can be employed for identifying andquantifying such Saccharomyces species.

BACKGROUND OF THE INVENTION

The identification and classification of the yeast genus Saccharomycesis of practical importance because some species are used in industriessuch as the beer, wine, baking and in biotechnology industries.Moreover, certain medical disorders are caused by yeast infections andappropriate treatment depends upon proper identification andclassification of the species.

For example, excessive colonization of the intestine with yeasts cancreate large amounts of alcohol (gastrointestinal alcohol fermentation).This condition, called "alcohol autointoxication syndrome" or"autobrewery syndrome," can lead to painful disturbances in the healthof patients. The ability to readily identify the causative agent of sucha condition would be valuable.

Also, it is known that following infection with S. cerevisiae,immunosuppressed patients can contract a fungemia, a sometimes fataldisorder. Rapid and unambiguous identification of the involved yeastscould contribute to speedy therapy.

Further, the ability to differentiate various types of yeasts could beused to identify yeasts more rapidly in investigations of the typeundertaken by Preller et al. J. Air Waste Managm. Ass. 39: 1094-1097(1989). There, chronic, non-specific pulmonary disorders frequentlyappeared in the inhabitants of a town located near a yeast productionplant and a penicillin production plant. In order to evaluate whichyeasts were linked causally with this disease symptom, investigatorsdetermined the number of S. cerevisiae particles per cubic meter of airin region between the yeast production plant and the town. A rapid testwould have permitted speedy identification of S. cerevisiae, and wouldhave distinguished S. cerevisiae from other yeast species, such as, forexample, Rhodotorula, which is frequently present in the air.

According to J. P. van der Walt, Genus 16: 555-718 (1970), 41 speciesare assigned to the genus Saccharomyces. However, many of these specieshave been assigned to other genera and renaming has taken place withinthe Saccharomyces genus. Currently, ten species are accepted within theSaccharomyces genus. See Barnett, J. A. Yeast 8: 1-23 (1992). These tenSaccharomyces species are: S. bayanus, S. castellii, S. cerevisiae, S.dairensis, S. exiguus, S. kluyveri, S. paradoxus, S. pastorianus, S.servazzii, and S. unisporus.

Moreover, in the future this classification likely will change becausethe rules and criteria by which taxonomic categorization of yeastspecies is undertaken are being changed. For example, one could arguethat the species S. bayanus, S. paradoxus and S. pastorianus are notindependent species but rather are varieties of S. cerevisiae.

Pursuant to Naumov et al. Yeast 8: 599-612 (1992), the followingterminology is used for describing the present invention: Saccharomycescerevisiae, Saccharomyces bayanus, Saccharomyces paradoxus andSaccharomyces pastorianus are combined as "Saccharomyces sensu stricto,"in contrast to "Saccharomyces sensu lato", which is used to designateSaccharomyces castellii, Saccharomyces dairensis, Saccharomyces exiguus,Saccharomyces kluyveri, Saccharomyces unisporus and Saccharomycesservazzii. The yeast species are classified taxonomically first andforemost by morphological and physiological criteria (such as, forexample, the utilization of carbon sources, dependence on growthfactors, utilization of nitrogen sources, and the like) and also bymating behavior. DNA analysis also is used increasingly for determiningrelationships between yeast genera and yeast species.

On the other hand, immunological methods are only rarely employed fordifferentiating yeast species. One reason for this is that antibodieswhich are directed against cell wall-specific antigens cross-react to alarge extent, both within a genus and beyond genera. The reason for thecross-reactivity within the genus Saccharomyces is that the cellwall-associated glycoproteins carry carbohydrate side groups which aremade up of mannose chains that do not differ, or differ slightly, fromspecies to species. There is also such a high degree of structuralhomology at the level of the polysaccharides (mannans and glucans) thatany differentiation using antibodies would seem to be very difficult toachieve.

The difficulty in obtaining antibodies which are specific for aparticular yeast species, in particular S. cerevisiae, is evident, forexample, from Kumar et al. Infection and Immunity 1840 (1985), 806-812).The authors report that antibodies which were in each case prepared inrabbits against a representative of the species Histoplasma capsulatum,Candida albicans or Saccharomyces cerevisiae in each case alsocross-react with the antigens of the other two species.

Consequently, a need exists for antibodies which react specifically withone or a few yeast species and thereby render it possible to use theseantibodies to differentiate these yeast species from others. Suchantibodies would be useful in certain foodstuff industries, such as thebeer and wine industries, in the medical diagnostic industry and in thepharmaceutical industry for quality control purposes.

For example, monoclonal antibodies specific for Saccharomyces sensustricto would be valuable in monitoring the culturing of S. cerevisiae(syn. S. boulardii) for use in drugs for treating diarrhea or forpreparing recombinant proteins of S. cerevisiae. Such monoclonalantibodies also would be useful for diagnosing conditions such as"alcohol autointoxication syndrome," fungemia, and non-specificpulmonary disorders.

SUMMARY OF THE INVENTION

Thus, an object of the present invention is the preparation ofantibodies or fragments thereof which specifically recognize the yeastspecies Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomycesparadoxus and Saccharomyces pastorianus, collectively referred to asSaccharomyces sensu stricto.

More specifically, the present invention relates to the monoclonalantibody Mab 92-276/018, or fragments thereof, from the hybridoma havingthe deposition number DSM ACC2126. Hereinafter this monoclonal antibodyis referred to as "Mab DSM ACC2126" or "Mab 92-276/018."

The invention also relates to epitopes or immunogenic moieties fromSaccharomyces sensu stricto, preferably from Saccharomyces cerevisiae,to which Mab 92-276/018 binds.

The invention further relates to antibodies or fragments thereof, whichbind to epitopes or immunogenic moieties of Saccharomyces sensu stricto,preferably Saccharomyces cerevisiae, and which possess the sameantigenic specificity as Mab 92-276/018.

In another embodiment, the invention relates to a hybridoma cell linethat secretes Mab 92-276/018 or secretes a monoclonal antibody whichpossesses the same antigenic specificity of Mab 92-276/018.

In another embodiment the invention relates to a diagnostic agent fordetecting Saccharomyces sensu stricto using the above describedantibodies or fragments thereof and a detectable label.

The present invention additionally relates to a diagnostic agent fordetecting antibodies against Saccharomyces sensu stricto, whichdiagnostic agent comprises (i) epitopes or immunogenic moieties fromSaccharomyces sensu stricto, preferably Saccharomyces cerevisiae, towhich the monoclonal antibody Mab 92-276/018 binds and (ii) a detectablelabel.

The invention further relates to a process for preparing monoclonalantibodies involving the isolation of immunogenic moieties fromSaccharomyces sensu stricto, preferably Saccharomyces cerevisiae, whichreact with the Mab 92-276/018; the immunization of a suitable host withthese isolated antigens or immunogenic moieties; and the selection ofthe resulting monoclonal antibodies that are specific for Saccharomycessensu stricto.

The present invention also relates to assays for identifying orquantifying Saccharomyces sensu stricto in a sample, wherein the sampleis brought into contact with one of the above-described Saccharomycessensu stricto specific antibodies and the resultant formation ofantigen/antibody complexes is then measured using suitable methods.

The invention also relates to assays for detecting antibodies againstSaccharomyces sensu stricto in a sample, wherein the sample is broughtinto contact with the above-described epitopes or immunogenic moietiesfrom Saccharomyces sensu stricto and the formation of resultantantigen/antibody complexes is then measured using suitable methods.

In yet another embodiment, the invention relates to a kit fordetermining the presence of Saccharomyces sensu stricto in a samplecomprising the above described monoclonal antibodies or fragmentsthereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to antibodies, and fragments thereof,specific for Saccharomyces sensu stricto. Exemplary of a such anantibody within the present invention is monoclonal antibody 92-276/018(DSM ACC2126). This antibody is directed against a cell wall-associatedantigen gp-200. Mab 92-276/018 is specific for Saccharomyces sensustricto, which includes S. paradoxus, S. pastorianus, S. bayanus and S.cerivisiae, as shown in the examples below.

The antibodies according to the present invention includes polyclonaland monoclonal antibodies. Monoclonal antibodies include any naturallyor non-naturally occurring polypeptide having the binding specificity ofMab 92-276/018, that is, a polypeptide which binds to the same epitopeon Saccharomyces sensu stricto, preferably S. cerivisiae, to which bindsMab 92-276/018. Examples of such polypeptides include a half antibodymolecule (a single heavy:light chain pair) or a fragment, such as theunivalent fragments Fab or Fab' and the divalent fragment F(ab')2 ("FAB"meaning fragment antigen binding), that possess the same specificity forbinding Saccharomyces sensu stricto, preferably S. cerivisiae, as Mab92-276/018.

A fragment, according to the present invention may also be a singlechain Fv fragment produced by methods well known in the art. See Skerraet al. Science 240: 1038-1041 (1988) and King et al. Biochem. J. 290:723-729 (1991), each of which is incorporated by reference. Themonoclonal antibodies of the present invention also include anon-peptide compound which is a "mimetic," i.e. a compound that mimicsthe epitope binding site of Mab 92-276/018 but that is water soluble,resistant to proteolysis and non-immunogenic. Conformationallyrestricted cyclic organic peptides which mimic Mab 92-276/018 can beproduced in accordance with methods well known to the skilled artisan.See e.g., Sargovi, et al., Science 253: 792-795 (1991), herebyincorporated by reference.

The terms "epitope" and "immunogenic moieties" as used in describingthis invention, include any determinant responsible for the specificinteraction with an antibody molecule. Epitopic determinants usuallyconsist of chemically active surface groupings of molecules such asamino acids or sugar side chains and have specific three-dimensionalstructural characteristics, as well as specific charge characteristics.

The monoclonal antibodies of the present invention also includemonoclonal antibody conjugates, which are for example, enzymes such ashorseradish peroxidase, alkaline phosphatase and β-D-galactosidase andfluorescent markers, such as fluorescein, fluorochrome, rhodamine andthe like. In such conjugates, the monoclonal antibody is bound to theenzyme or marker directly or by way of a spacer or linker group, such asethylenediamine-tetraacetatic acid (EDTA) or the like.

Polyclonal and monoclonal antibodies can be produced in various waysusing techniques well-understood by those having ordinary skill in theart. Details of these techniques are described in Antibodies: ALaboratory Manual, Harlow et al. Cold Spring Harbor Publications, p. 726(1988), which is hereby incorporated by reference.

Generally, the monoclonal antibodies of the present invention areprepared by immunizing BALB/c mice subcutaneously and intraperitoneallywith S. cerivisiae protein gp200, as described below in Examples 1 and2. Spleens from the immunized mice are fused with SP2/0 myeloma cellsand hybridomas secreting antibodies against gp200 are detected using anenzyme assay.

Thus, in one embodiment, the present invention relates to a hybridoma.This hybridoma secretes a monoclonal antibody that is specific forSaccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces paradoxusand Saccharomyces pastorianus. In particular, the hybridoma of thepresent invention secretes murine monoclonal antibody, Mab 92-276/018,which was deposited with the Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSM) (German Collection of Microorganisms and CellCultures), Mascheroder Weg 1 B, 38124 Braunschweig, on Apr. 22, 1993under the number DSM ACC2126. The hybridoma cell lines of the presentinvention are genetically stable, secrete monoclonal antibodies of theinvention and can be activated by standard techniques.

The monoclonal antibodies and fragments thereof according to thisinvention are multiplied according to in vitro and in vivo methodswell-known in the art. Multiplication in vitro is carried out insuitable culture media such as Dulbecco's Modified Eagle Medium or RPMI1640 medium, optionally replenished by a mammalian serum such as fetalcalf serum or trace elements and growth-sustaining supplements, e.g.,feeder cells, such as normal mouse peritoneal exudate cells, spleencells, bone marrow macrophages or the like. In vitro production providesrelatively pure antibody preparations and allows scale-up to give largeamounts of the desired antibodies. Techniques for large scale hybridomacultivation under tissue culture conditions are known in the art andinclude homogenous suspension culture, e.g., in an airlift reactor or ina continuous stirrer reactor or immobilized or entrapped cell culture.

Large amounts of the monoclonal antibody of the present invention alsomay be obtained by multiplying hybridoma cells in vivo. Cell clones areinjected into mammals which are histocompatible with the parent cells,e.g. syngeneic mice, to cause growth of antibody producing tumors.Optionally, the animals are primed with hydrocarbon, especially oilssuch as pristaine (tetramethylpentadecane) prior to injection. After oneto three weeks, the desired monoclonal antibody is recovered from thebody fluid of the mammal.

In accordance with the present invention, fragments of the monoclonalantibody of the invention can be obtained form the monoclonal antibodyproduced as described above and in Example 2, by methods which includedigestion with enzymes such as pepsin or papain and/or cleavage ofdisulfide bonds by chemical reduction. Alternatively, monoclonalantibody fragments encompassed by the present invention can besynthesized using an automated peptide synthesizer as supplied byApplied Biosystems, Multiple Peptide Systems, etc. or they may beproduced manually, using techniques well-known in the art. See Geysen,et al. J. Immun. Methods 102: 259-274 (1978), hereby incorporated byreference.

The monoclonal conjugates of the present invention are prepared bymethods known in the art, e.g., by reacting a monoclonal antibodyprepared as described above with, for instance, an enzyme in thepresence of a coupling agent such as glutaraldehyde or periodate.Conjugates with fluorescein markers are prepared in the presence ofthese coupling agents or by reaction with an isothiocyanate. Conjugateswith metal chelates are similarly produced.

Radioactively labeled monoclonal antibodies of the present invention areproduced according to well-known methods in the art. For instance,monoclonal antibodies can be iodinated by contact with sodium orpotassium iodide and a chemical oxidizing agent, such aslactoperoxidase. Monoclonal antibodies according to the invention may belabeled with technetium-99m by ligand exchange process, for example, byreducing pertechnate with stannous solution, chelating the reducedtechnetium onto a Sephadex column and applying the antibody to thiscolumn or by direct labelling techniques, e.g., by incubatingpertechnate, a reducing agent such as SNCl2, a buffer solution such assodium-potassium phthalate solution, and the antibody. Radionuclides canbe bound to an antibody either directly or indirectly by using anintermediary functional group. Intermediary functional groups which areoften used to bind radioisotopes which exist as metallic ions toantibody are diethylene-triaminepentaacetic acid (DTPA) andethylenediaminetetracetic acid (EDTA). Examples of metallic ions suitable foruse in the invention are ^(99m) Tc, ¹²³ I, ¹³¹ I, ¹¹¹ In, ¹³¹ I, ⁹⁷ Ru,⁶⁷ Cu, ⁶⁷ Ga, ¹²⁵ I, ⁶⁸ Ga, ⁷² As, ⁸⁹ Zr, and ²⁰¹ Tl. In accordance withthis invention, the monoclonal antibody or fragment thereof may belabeled by any of several techniques known to the art. See, e.g., Wagneret al. J. Nucl. Med. 20: 428 (1979) and Saha et al., J. Nucl. Med. 6:542 (1976), hereby incorporated by reference.

Thus, in one embodiment, the invention relates to a diagnostic agentcomprising the above describe monoclonal antibody and a label for thedetection of S. sensu stricto. In another embodiment, the inventionrelates to a diagnostic agent for the detection of antibodies against S.sensu stricto comprising an epitope of S. sensu stricto and a detectablelabel.

The invention also relates to various immunological detection systems,such as Western blot, immunofluorescence test, enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescenceimmunoassay (FIA). Indeed, the invention includes in vitro assays forthe detection of S. stricto sensu in whatever kind of sample it mayoccur, such samples including fluid, semi-fluid or tissue samples, usingthe monoclonal antibody or fragment thereof of the invention. The assaycan be a competitive of sandwich assay, or any assay well-known to theartisan which depends on the formation of an antibody-antigen immunecomplex. For purposes of this invention, the monoclonal antibody orfragments thereof of the present invention can be immobilized orlabeled. Many carriers are known to the skilled artisan to which themonoclonal antibody or fragments thereof of the present invention can bebound for immobilization. Where required, derivatization techniques canbe used for immobilizing the monoclonal antibody or fragment thereof ona substrate. Well-known carriers include glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amylases, natural andmodified celluloses etc. The carrier can be either soluble or insoluble.Immunoassays encompassed by the present invention include, but are notlimited to those described in U.S. Pat. Nos. 4,367,110 (doublemonoclonal antibody sandwich assay); Wide et al., Kirkham and Hunger,eds. Radioimmunoassay Methods, E. and S. Livingstone, Edinburgh (1970):U.S. Pat. No. 4,452,901 (Western Blot); Brown et al., J. Biol. Chem.255: 4980-4983 (1980) (immunoprecipitation of labeled ligand); andBrooks et al. , Clin. Exp. Immunol. 39: 477 (1980)(immunocytochemistry).

The in vitro assays of the present invention also include the use ofepitopes or immunogenic moieties of S. sensu stricto in the detection ofantibodies against S. sensu stricto. For example epitopes or immunogenicmoieties may be bound to a solid substrate and used in assays todetermine the effects of modifications to known monoclonal antibodieshaving the same binding affinity as Mab DSM ACC2126. Another assayinvolves the detection of monoclonal antibodies in samples which competewith Mab DSM ACC2126 in binding to S. sensu stricto or a polypeptidepresenting the epitope of the invention. In that way, the presence ofantibodies, for example from a patient sample (e.g., blood, urine,etc.), can be determined without the need for the epitope mappingdiscussed below.

From the disclosure provided herein, a skilled artisan readily couldcharacterize, i.e., map, the epitope or immunogenic moieties of S. sensustricto responsible for binding monoclonal antibodies having the samebinding affinity as Mab DSM ACC2126. In this regard, see Example 8.Indeed, monoclonal antibody mapping analysis techniques are well-knownin the art, see e.g. Regenmortal, Immunology Today 10: 266-72 (1989) andBerzofsky et al., Immunological Reviews 98: 9-52 (1987), and may includecomputer predictions of secondary structure and antigenicity to indicatethe possible location of epitopes, particularly as conserved regions ofthe protein may be of structural or functional significance. Furtherinvestigations also involve crossblocking of monoclonal antibodies todefine classes of monoclonal antibodies that bind to identical oroverlapping regions. The predicted epitope is confirmed using clonedoligonucleotides or synthetic peptides. Important residues are thenidentified by mutagenesis or cloning of incorrect oligonucleotides or byusing incorrect synthetic peptides. Alternatively, epitope mapping maybe carried out according to the present invention by usinghomolog-scanning mutagenesis in conjunction with secondary structureanalyses. See Cunningham, et al., Science 243: 1330-1336 (1989). Thisinvolves the synthesis of many small peptides that completely span theprotein and allows identification of many small continuous epitopes of aprotein or fragment. Another method of epitope mapping involves therandom cloning of very small fragment of DNA encoding parts of theprotein and screening each clone obtained with each available monoclonalantibody. See generally, Immunochemical Protocols, Manson, M. ed., vol.10, Humana Press, Totowa, N.J. pp. 105-116 (1992).

Thus, in one embodiment, the appropriate sample of S. sensu stricto isdigested into polypeptide fragments and binding assays using, forexample, Mab DSM ACC2126, are performed to locate the polypeptidefragment responsible for binding the Mab. Subsequent sequencing of thefragment would provide the amino acid sequence of the epitope. SeeManson, supra.

The polypeptide presenting the epitope then could be reproduced usingstandard cloning and expression techniques using procaryotic oreucaryotic hosts, depending upon the desirability of glycosylation ofthe epitope. For example, one could synthesize DNA encoding the aminoacid sequence of the epitope, using preferred codons for the host. Thelength of the amino acid sequence used would depend upon theconformational requirements of the epitope, i.e., the length of sequencerequired to provide the conformation that yields acceptable binding toMab DSM ACC2126. The DNA then could be inserted into a plasmid vectorthat in turn could be used to transfect the desired host. Upon culturingin appropriate growth medium, the epitope would be produced.

Skilled artisans readily may appreciate the many acceptable variationsof the foregoing processes, as well as other ways in which the epitopecould be characterized and reproduced by recombinant or non-recombinantmeans.

The monoclonal antibodies or fragments thereof of the present inventionare suitable for use in a kit. Such a kit may comprise a receptaclebeing compartmentalized to receive one or more containers, such asvials, tubes and the like, such containers holding separate elements ofthe invention. For example, one container may contain a first antibodybound to an insoluble or partly soluble carrier. A second container maycontain soluble, detectably-labeled second antibody, in lyophilized formor in solution. The receptacle may also contain a third containerholding a detectably labeled third antibody in lyophilized form or insolution. A kit of this nature can be used in the sandwich assay of theinvention.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following examples are merely illustrative andnot limitative of the remainder of the disclosure in any way whatsoever.

EXAMPLE 1 Isolation of gp200

S. cerevisiae gp200 was isolated according to the method of Heelan etal. Immuno. Lett. 28: 181-186 (1991). Specifically, S. cerevisiae wasgrown aerobically for 48 hours on Sabouraud agar (Oxoid) at 37° C.Soluble extracts (Sacc) were prepared as described previously inMcKenzie et al. Gut 31: 536 (1990) and Barnes et al. Int. Arch. AllergyAppl. Immuno. 92: 9 (1990). Cells were harvested, washed twice in 0.15Msaline, resuspended in saline (1:10, v/v) after centrifugation at 2000×gfor 10 minutes), the supernatant removed, sterile-filtered (0.2 μm) andstored in aliquots at -20° C. The protein concentration was determinedby Bio-Rad protein assay.

Sera from CD patients containing IgG and IgA isotype-specific antiSaccantibodies were determined by enzyme-linked immunosorbent assay (ELISA)as previously described in Barnes, supra. Briefly, CD sera (1:100dilution) were added to microtitre plates coated with Sacc (10 μ/ml),followed by peroxidase-conjugated rabbit anti-human γ or anti-α chain(1:1000). Sera with binding indices >3.0 were regarded as anti-Saccantibody-positive. Barnes, supra. Control anti-Sacc antibody-negativesera were obtained from blood donors Barnes, supra.

Peroxidase-conjugated rabbit IgG anti-human Ig, anti-human gamma andanti-human alpha heavy chains, as well as peroxidase-conjugated swine(IgG anti-rabbit Ig, were obtained from Dako Ltd. (High Wycombe). Rabbitantiserum to S. cerevisiae was obtained by repeated immunization withsoluble antigen (Sacc) in incomplete Freund's adjuvant: anti-Saccantibody activity, compared with pre-immune serum, was determined byELISA using peroxidase-conjugated swine anti-rabbit Ig.

Gels were run using the discontinuous buffer system of Laemmli, Nature227: 680 (1970) with a 3% stacking gel and 12% resolving gel. Sampleswere reduced by heating at 100° C. for 2 minutes in 25 mM dithiothreitol(DTT; Sigma). 20-μl samples (1:1 in sample buffer) of whole S.cerevisiae (1:10, v/v in saline) solubilized in 2% SDS by heating at100° C. for 2 minutes, or saline extract (i.e., Sacc) of S. cerevisiae(0.5 mg/ml), were applied to each gel. Gels were stained with Coomassiebrilliant blue or periodic acid Schiff's reagent (PAS). Towbin et al.Proc. Natl. Acad. Sci. USA 76: 4350 (1979)

Transfer of antigens from gels on to nitrocellulose sheets (GelmanSciences) was performed overnight in a Trans-Blot electrophoretictransfer cell (Bio-Rad). See Johnston et al. Immunochem. in Practice,Blackwell, Oxford (1987). After transfer, nitrocellulose strips werewashed twice in Tris-buffered saline (TBS), pH 7.4, for 15 minutes,followed by a 30-minute wash in 10% foetal calf serum (FCS)/TBS. Stripswere exposed to human sera containing anti-Sacc antibodies orantibody-negative control sera (diluted 1:100 in 10% FCS/TBS) for 2-3hours. Unbound primary antibody was removed by two 15-minute washes inTBS, after which peroxidase-conjugated rabbit antihuman Ig, antiIgG oranti-IgA (diluted 1:500 in 10% FCS/TBS) was added for 1-2 hours.Following removal of unbound antibody with two 5-minute washes indistilled water, 0.1% 4-chloro-1-naphthol (Sigma) in TBS with fresh0.02% H₂ O₂ was used as substrate.

This was performed after transfer to nitrocellulose strips as describedin Woodward et al., J. Immunol. Methods 78: 143 (1985). All strips wererinsed with 50 mM sodium acetate buffer, pH 4.5, after which controlstrips were incubated in the same buffer for 1 hour. Test strips wereexposed to varying concentrations of sodium m-periodate, from 0.1 to 50mM in pH 4.5 acetate buffer, for 1 hour in the dark at 23° C. Controland test strips were then rinsed with sodium acetate buffer, exposed to50 mM sodium borohydride in phosphate-buffered saline, pH 7.4, for 30minutes at 23° C., followed by a further 5 minute wash in TBS. Blottingwas performed using rabbit anti-Sacc serum and pre-immune rabbit serum(NRS) (both 1:100 in 10/% FCS/TBS) followed, after washing, byperoxidase-conjugated swine anti-rabbit Ig (1:500).

SDS-PAGE of a saline extract (Sacc) of S. cerevisiae showed a singleband of apparent size 200 kDa under reducing and non-reducingconditions. This band always stained with Coomassie blue, althoughweakly, and strongly with PAS. This 200 kDa glycoprotein migrated inidentical position to a single glycoprotein band in SDS-solubilizedwhose S. cerevisiae.

Following SDS-PAGE of a saline extract of S. cerevisiae (Sacc) andSDS-solubilized S. cerevisiae, nitrocellulose strips were blotted with 8anti-Sacc antibody-positive sera and two antibody-negative sera from CDpatients. Eight out of 8 anti-Sacc antibody-positive sera reacted with asingle band of apparent size 200 kDa present in both whole S. cerevisiaeand Sacc. Confirmation of antibody-binding with the 200 kDa band in Saccwas obtained using eight sera containing IgG or IgA isotype-specificanti-Sacc antibodies. Reactivity with the 200 kDa band was obtained onlywith IgG and IgA specific anti-Sacc antibodies; control sera showednegative binding. Rabbit anti-Sacc serum showed identical reactivitywith this 200 kDa band, compared with pre-immune rabbit serum which wasnon-reactive.

Confirmation of the glycoprotein composition of gp200 was obtained byperiodate treatment of Sacc antigen, followed by blotting with therabbit anti-Sacc serum. Increasing concentrations, up to 50 mM, ofperiodate caused increasing, but not complete, loss of immunoreactivitywith the 200 kDa band.

EXAMPLE 2 Preparation of Monoclonal Antibodies Against the Glycoproteingp200 From Saccharomyces cerevisiae

Balb/c mice which were six to eight weeks old were immunized with aglycoprotein (gp200), as described above. The gp200 can be isolatedrapidly and simply by culturing S. cerevisiae (J. Sainsbury, PLC)overnight in YPD medium. The cells are harvested, washed twice in waterand resuspended in 10 times the volume of water. The cell suspension isincubated for 1 hour in a water bath at 100° C. The cell preparation isthen centrifuged for 5 minutes at 5000 rpm in a bench centrifuge(Varifuge 3.2 RS) and the supernatant concentrated approximately 3 timesusing a Centricon 100k filtration system (tiltron).

Then, each mouse was injected subcutaneously with approximately 50 μg ofthe glycoprotein emulsified in complete Freund's adjuvant and, in asecond instance, intraperitoneally. A second and a third immunizationwithout adjuvant were in each case carried out four to eight weekslater. Immediately before the actual fusion, the experimental animalswere additionally boosted intravenously on four days in succession. Onthe day of the fusion, the spleens were removed under sterile conditionsand suspended to form individual cells. By means of fusing 10⁸ spleencells with 2×10⁷ cells of the myeloma cell line SP 2/0 or cell line×63Ag 8653 (J. Immunol. 173: 1548-1550, 1979!), hybrid cells were producedwhich were subsequently sown in a selection medium (Dulbecco's minimalessential medium, DMEM, supplemented with 20% fetal calf serum FCS, 0.1mM hypoxanthine, 0.4 mM aminopterin and 16 mM thymidine) on cultureplates having 24 wells (from Costar) at a concentration of 10⁶cells/well. Two to three weeks later, individual cell colonies wereisolated from the wells and in each case transferred into a well of anew culture plate.

After a further two to three days, the culture supernatants werescreened for the presence of gp200-specific antibodies using an enzymeimmunoassay. The supernatants were incubated on gp200-coatedmicrotitration plates and any specific antibodies which were presentwere detected by an anti-mouse peroxidase/immunoglobulin conjugatereaction. Positive cell lines were cultured up and, after testing forthe absence of mycoplasmas, were frozen in liquid nitrogen. In parallelwith this, positive cell lines were cloned using a micromanipulator.Suitable clones were then multiplied in cell culture (mass culture inroller bottles). Purification was carried out using ammonium sulfateprecipitation and protein A chromatography. Purity was tested by meansof HPLC and gel electrophoresis and the immunoglobulin classesdetermined by Ouchterlony immunodiffusion.

EXAMPLE 3 Selection of Monoclonal Antibodies

Hybridoma supernatants are tested for their reactivity in Western blotsusing antigen from Saccharomyces cerevisiae and from Schizosaccharomycespombe as a comparison.

For this purpose, S. cerevisiae (VLSF 07158) and S. pombe (CBS 1043)were cultured for 3 days, at 30° C. and at 180 rpm, in YPD medium (1%yeast extract, 2% peptone, 2% glucose) in 250 ml conical flasks on ashaking apparatus. After centrifuging 10 ml of culture both in eachcase, the cells were harvested, taken up in 1 ml of 10 mM Tris buffer(pH 7.5), provided with glass beads, and disrupted in a glass bead mill.Subsequently, the homogenate was heated in accordance with Laemmli,supra., fractionated in a 10% SDS polyacrylamide gel and transferred tonitrocellulose membranes (Burnette, Anal. Biochem., 112: 195-203 1981!).The nitrocellulose strips were then incubated in Tris buffers (10 mMTris HCl, pH 7.5, 150 mM Nacl, 1% gelatin, 4% tween 20) which in eachcase contained monoclonal antibodies, at a concentration 6fapproximately 10 μg/ml, from nine different hybridoma supernatants.Anti-mouse antibodies which were coupled to alkaline phosphatase wereused as the conjugate. Fast Blue B salt (from Serva, Heidelberg) andNaphtol AS-MX Phosphate (from Sigma Chemie, Munich) were used assubstrates. Rainbow® protein molecular weight markers (Amersham BuchlerGmbH, Braunschweig) were additionally loaded onto the SDS polyacrylamidegel for determining the relative molecular mass.

All nine antibody specificities recognized antigens from S. cerevisiaein the Western blot.

Of nine supernatants tested, four were only very weakly positive:

-92-285/18

-92-285/27

-92-285/29

-92-275/02

Four supernatants exhibited quite good reactivity:

-92-285/5

-92-285/10

-92-285/15

-92-285/16

The strongest reactivity was shown by the supernatant -92-276/018.

By contrast, none of the nine supernatants recognized an antigen from S.pombe. This was judged to be the criterion for a specificimmunoreactivity of the monoclonal antibodies.

In subsequent work, the reactivity of the monoclonal antibody 92-276/018was investigated in more detail.

EXAMPLE 4 Specificity of Mab 92-276/018

The specificity of the monoclonal antibody 92-276/018 (DSM ACC2126) wasanalyzed in Western blots on the one hand and on intact yeast cells inimmunofluorescence tests on the other.

Immunofluorescence on intact yeast cells using monoclonal antibodies wascarried out as follows. The yeasts were cultured in media as describedfor the Western blot analyses. 10 μl of a yeast cell suspension were ineach case introduced into the wells of immunofluorescence glass plates.After 1 hour of incubation at RT, the supernatant was sucked off and thecells were fixed by adding acetone cooled to 4° C. The plates were thenrinsed with water and dried. 10 μl of the antibody solution (100 μg/ml)were pipetted into each well. After the plates had been incubated at 37°C. in a moist chamber for one hour, the supernatant was removed, theplates were washed three times, and FITC-labeled anti-mouseimmunoglobulin conjugate was added.

Microscopic analysis was carried out after the plates had been incubatedfor one hour and then washed.

Mab 92-276/018 was examined with regard to its reactivity with differentyeast species. The different yeast species were obtained from thefollowing institutions: Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSM) (German Collection of Microorganisms and CellCultures), Braunschweig, Federal Republic of Germany; Centraalbureauvoor Schimmelcultures (CBS) (Central Office for Mould Cultures),Baarn-Delft, Netherlands; Versuchs- und Lehranstalt furSpiritusfabrikation und Fermentationstechnologie in Berlin (VLSF)(Experimental and Educational Institute for Spirit Production andFermentation Technology in Berlin), Berlin, Federal Republic of Germany;Institut fur Mikrobiologie und Weinforschung (IMW) (Institute forMicrobiology and Wine Research) of the Johannes Gutenberg University,Mainz, Federal Republic of Germany; Yeast Genetic Stock Center (YGSC),Berkeley, USA and J. Sainsbury PLC, London, England. In addition tothis, some yeast strains were obtained from research institutes, whichstrains only have strain designations which are cited in the literaturebut do not conform to the nomenclature system of any official institute.The following yeast species were employed:

    ______________________________________    Yarrowia lipolytica    DSM 1345    Debaromyces hansenii   DSM 3428    Hanseniaspora quillierinondii                           DSM 3432    Schwanniomyces occidentalis                           DSM 3451    Lipomyces starkeyi     DSM 70295    Pichia pastori         DSM 70382    Zyqosaccharcznyces bailii                           DSM 70492    Saccharomyccdes ludwiqii                           DSM 3447    Torulaspora delbrueckii                           DSM 70504    Torulaspora pretcriensis                           DSM 70525    Hanseniaspora uvarum   IMW 473    Schizosaccharoiuyces pombe                           CBS 1043    Dekkera anomala        CBS 4210    Kluyveromyces marxianus                           CBS 369    Pachysolen tannophilus CBS 4045    Candida boidinii       CBS 5777    Candida albicans       CBS 2730    ______________________________________

In this collection of yeast species, four species are also representedwhich were previously assigned to the genus Saccharomyces:

    ______________________________________    Kluyveromyces marxianus                     ex Saccharomyces fragilis                     ex Saccharomyces lactis    Zygosaccharomyces bailii                     ex Saccharomyces elegans    Torulaspora delbrueckii                     ex Saccharomyces fermentati    Torulaspora pretoriensis                     ex Saccharomyces pretoriensis    ______________________________________

These four yeast species, which at the time were assigned to the genusSaccharomyces on the basis of morphological and physiological criteria,and which exhibit a high degree of similarity with the genusSaccharomyces as regards taxonomic criteria, can thus be clearlydelimited immunodiagnostically from Saccharomyces cerevisiae using themonoclonal antibody 92-276/018.

None of these yeast species from different families reacted with themonoclonal antibody 92-276/018, either in the immunofluorescence test orin Western blot analyses.

EXAMPLE 5 Further Reactivity of Mab 92-276/018 (Wild)

Further work examined the reactivity of the monoclonal antibody92-276/018 with different wild isolates of the species S. cerevisiae.

The following strains of S. cerevisiae were employed:

    ______________________________________    S. cerevisiae DSM 70471                         (ex. S. ellipsoideus)    S. cerevisiae DSM 70478                         (ex. S. chevallieri)    S. cerevisiae DSM 70487                         (ex. S. diastaticus)    S. cerevisiae DSM 70514                         (ex. S. italicus)    S. cerevisiae CBS 5926                         (ex. S. boulardii)    S. cerevisiae IMW 116    S. cerevisiae IMW 188    S. cerevisiae IMW 25    S. cerevisiae IMW 92    S. cerevisiae VLSF 07158    S. cerevisiae J. Sainsbury, PLC    S. cerevisiae YGSC X2180 1A    ______________________________________

All these strains reacted with the monoclonal antibody, both in theWestern blot and in the immunofluorescence test. The first four strainsexamined had previously been assigned to different Saccharomyces specieswhich are now no longer universally accepted, and all of them have beenadded to the species S. cerevisiae.

EXAMPLE 6 Further Reactivity of Mab 92-276/018 (Lab)

Further work examined the reactivity of the monoclonal antibody withdifferent laboratory strains of the species S. cerevisiae. The followingtable lists the strains examined, their genetic markers and theirreactivity with the monoclonal antibody.

                  TABLE 1    ______________________________________    Reactivity of the monoclonal antibody 92-276/018 with    Saccharomyces cerevisiae laboratory strains                              Immuno-  Immuno-    Strain  Genetic Markers   blot     fluorescence    ______________________________________    C13ABYS86            ura3-2,leu2,his,pra1,prh1,                              +        +            prc1,cps1    HT 393  ura3,leu2,pra1,prb1,prc1,cps1,                              +        +            pre1    79      leu2,trp1         +        +    S150-2B leu2-3,leu2-112,ura3-52,                              +        +            tp1-289,his3Δ1    TY 4    leu2,ura3,ts1     +        +    4STLU   leu2,ura3,ts1,srb1                              +        +    CGY 1465            leu2,ura3,ssc1/pmr1                              +        +    A 258   can1,leu2,his4,met14,trp1,ura3,                              +        +            ose1,SUC2    A 259   can1,leu2,his4,met14,trp1,ura3,                              +        +            OSE1,SUC2    ______________________________________

All the strains reacted, independently of the genetic markers. S.cerevisiae strains having an altered cell-wall structure or proteindeletions in the periplasmic space due to genetic mutations, such as,for example, 4STLU, CGY 1465, TY 4 and A 258, were also recognized bythe monoclonal antibody.

The laboratory strains of S. cerevisiae listed here are predominantlyused for recombinant DNA purposes, and serve, in particular, as hostcells for expressing heterologous proteins. These strains are freelyavailable and are described in the literature with regard to theirmarkers and possible uses. The specific references are:

    ______________________________________    A 258 and A 259:              Sakai et al., Genetics 119, pp. 499-506  1988!.    CGY 1465: Rudolph et al., Cell 58, pp. 133-145  1989!.    C13ABYS86 and              Heinemeyer el al., EMBO J. 10, pp. 535-562  1991!.    HT 393:    4STLU and TY 4:              Waltschewa et al., Yeast 5, pp. 313-320  1989!.    S150-2B:  Baldari et al., EMBO J. 6, pp. 229-234  1987!.    79:       Price et al., Gene 55, pp. 287-293  1987!.    ______________________________________

EXAMPLE 7 Reactivity of 92-276/018 With Saccharomyces Species Other ThanS. cerivisiae

According to Barnett, ten species are nowadays assigned to the genusSaccharomyces. An investigation was next undertaken to determine whetherthe monoclonal antibody 92-276/018 reacts with the other nine species inaddition to S. cerevisiae.

The monoclonal antibody 92-276/018 showed no reaction in a Western blotand in an immunofluorescence test with the following Saccharomycesspecies:

    ______________________________________           S. exigus     CBS 134           S. dairensis  CBS 421           S. unisporus  CBS 398           S. servazzii  CBS 4311           S. castelli   CBS 4309           S. kluyveri   CBS 2861    ______________________________________

However, the monoclonal antibody recognized the species:

    ______________________________________    S. bayanus           DSM 70412    S. bayanus           DSM 70511    S. bayanus           DSM 70547    S. pastorianus       DSM 6580    S. pastorianus       CBS 1260    S. paradoxus         CBS 406    S. paradoxus         CBS 2980    S. paradoxus         CBS 432    ______________________________________

It is evident, therefore, that antibodies against the gp200 from S.cerevisiae and, in particular, the monoclonal antibody 92-276/018,represent suitable agents for identifying the species S. cerevisiae or,alternatively, characterizing a yeast species other than S. cerevisiaeto show that this species cannot possibly be S. cerevisiae.

The fact that the monoclonal antibody 92-276/018 recognizes the speciesS. paradoxus, S. pastorianus and S. bayanus in addition to S. cerevisiaedoes not indicate lack of specificity. Extensive DNA studies haverevealed that S. paradoxus and S. bayanus exhibit an exceptionally highdegree of relatedness with S. cerevisiae (Naumov et al., Yeast, 8:599-612 1992!). In addition to this, the species S. pastorianus isconsidered to be a hybrid of the species S. cerevisiae and S. bayanus.This high degree of similarity between the species led Naumov et al. todescribe these Saccharomyces species as being sibling species of S.cerevisiae.

Because the underlying criteria for determining yeast species aresubject to variation and alteration with time, the species S. paradoxus,S. pastorianus and S. bayanus should not be regarded as independentspecies but rather as varieties of S. cerevisiae. However, even ifBarnett's classification of the species within the genus Saccharomycesis accepted as being valid, the monoclonal antibodies of the presentinvention are of great value because they identify a yeast isolate asbelonging to the genus Saccharomyces and exclude several species withinthis genus.

EXAMPLE 8 Analysis of Epitope

Applicants observed that if nitrocellulose paper onto which proteins ofS. cerevisiae cell extracts have been transferred from polyacrylamidegels is incubated in periodate solutions and the reactivity whichremains is subsequently investigated using the monoclonal antibody92-276/018, after a 30-minute incubation in the presence of 0.1 mMperiodate, the full reactivity is still present. By contrast, applicantsdetected no binding of the monoclonal antibody 92-276/018 when thenitrocellulose strips were incubated in 10 mM or 50 mM periodate.

The reactivity is strongly affected when the incubation of themonoclonal antibody with the nitrocellulose coated with S. cerevisiaeantigens is carried out in the presence of 100 mMmethyl-α-D-methylmannoside. By contrast, the reactivity is undiminishedas compared with the control, to which no sugars have been added, in thepresence of methyl-α-D-methylglucanoside.

Applicants conclude from these two findings that the antigenicdeterminants which are recognized by the monoclonal antibody 92-276/018probably represent carbohydrate structures, or represent peptidestructures whose affinity for the antibody is influenced by carbohydratesidechains.

In Western blot analyses, it is not only antigen which bands in SDS gelswith an apparent molecular weight of 200 kDa which is recognized by themonoclonal antibody 92-276/018 (Mab DSM ACC2126); rather, the specificreactivity extends over a relatively wide range from about 40 kDa to 200kDa. This indicates that mannoproteins or mannans are being recognizedwhich are very heterogeneous in their molecular weight. It is probablethat the monoclonal antibody recognizes a carbohydrate-specific epitopewhich can be a constituent of several glycoproteins or mannan structuresand that gp200 represents only one antigen of several in S. cerevisiaehaving such an epitope.

What is claimed is:
 1. An isolated and purified epitope fromSaccharomyces sensu stricto to which the monoclonal antibody 92-276/018binds.
 2. The epitope of claim 1 which is from Saccharomyces cerevisiae.3. A diagnostic agent for detecting antibodies against Saccharomycessensu stricto, said diagnostic agent comprising the epitope of claim 1labeled with a detectable label.
 4. An assay for detecting antibodiesagainst Saccharomyces sensu stricto in a sample comprising contactingsaid sample with the epitope of claim 1 and detecting the specificbinding of said antibody in said sample with said epitope.